The present invention relates in general to electron beam particle imaging, and more particularly, to a method of electron beam imaging of a specimen by combining images of an image sequence.
TEM (transmission electron microscopy) performance lags considerably behind its theoretical limit based on the physics of electron interactions with matter, especially in cases where low-dose imaging is required because of specimen sensitivity to damage by the electron beam. Multiple factors can reduce resolution and signal-to-noise ratio (SNR) in TEM images, including a number of factors related to electron microscopy instrumentation and the use of non-ideal electron detectors. Taken together, however, instrumentation factors explain only a small fraction of the gap between theoretically attainable and actual performance. Dynamic specimen processes, such as drift, beam-induced motion, charging, radiation damage, generally contribute a significant portion of the non-idealities that reduce overall system performance. Additionally, non-ideal electron detectors are a significant final factor explaining the gap between actual and theoretical performance in TEM.
Radiation damage to a specimen refers to the breakdown of the chemical structure of a specimen under observation as a result of interaction with the electron beam. Inelastic scattering of the electrons on the specimen causes excitation of specimen valence electrons resulting in radiation damage, which includes bond rupture, free radical formation, liberation of hydrogen atoms, changes in the physical properties of the specimen (e.g., density), and structural rearrangements. Generally, damage is cumulative, meaning that as the total exposure accumulates on the specimen, damage continues to increase, first affecting high-resolution features and then affecting lower-resolution features as exposure continues to accumulate. Cumulative damage may also depend on dose rate, meaning that 20 electrons per square Ångström delivered over a short period of time (e.g., one second) may cause more damage than the same number of electrons delivered over a longer period of time (e.g., five seconds).
Sensitive specimens, such as specimens of biological importance held in natural states of hydration (frozen in amorphous ice), are imaged in the electron microscope using both a low total electron exposure (total number of electrons used to form an image, e.g., 20 electrons per square Ångström on the specimen) and a low electron exposure rate (number of electrons per unit time, per unit area, used to illuminate a specimen for acquisition of an image, e.g., 10 electrons per square Ångström per second on the specimen). However, in such cryo-microscopy of biological specimens, dynamic specimen processes are particularly detrimental, causing either non-isotropic resolution loss (i.e., specimen drift) or overall degradation of the SNR in each image (e.g., beam-induced motion, charging, radiation damage, etc.). Moreover, such dynamic specimen processes are exasperated in the context of sequential imaging in a “movie” mode with extended electron beam exposure.
Photographic film or CCD-based electronic cameras used to record TEM images collect a single image representing the entire electron exposure of the specimen. A sequence of multiple images of a specimen may be acquired by either manually or automatically collecting multiple images of the specimen, one after another, with some amount of dead time between each image acquisition. Alternatively, CMOS-based electronic cameras can collect a sequence of images of the specimen, with negligible dead time between each image, so that an electron exposure is intrinsically fractionated into multiple images. In either case, a sequence of multiple images of a specimen may be combined (for example, by addition) to form a single static specimen image.
Recently, the inventors, among others, developed a TEM camera system based on a specially developed active pixel sensor (APS) for detection of electrons by direct bombardment on the sensor, called the Direct Detection Device (DDD). The DDD has significantly higher sensitivity and resolution than other electronic cameras and offers a large field of view similar to photographic film. The architecture of this DDD camera also allows for continuous streaming of full-resolution, full-frame images at up to 40 frames per second, with little or no dead time between consecutive frames.
Accordingly, there exists a need in the art for an improved imaging technique in comparison to the prior art.